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体外扩增DNA假文库的制备及高通量cDNA靶标扩增。

Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification.

作者信息

Frey Daniel, Kambach Christian, Steinmetz Michel O, Jaussi Rolf

机构信息

Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland.

出版信息

BMC Biotechnol. 2007 Jun 12;7:31. doi: 10.1186/1472-6750-7-31.

DOI:10.1186/1472-6750-7-31
PMID:17565673
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1894961/
Abstract

BACKGROUND

Many structural biology- and high-throughput laboratories experience the acquisition of multiple cDNAs from different sources as a rather time- and resource-consuming procedure. The techniques presented here solve these problems.

RESULTS

An advanced target cDNA amplification procedure employing RNA- or cDNA-derived pseudolibraries circumvents the usual DNA transfection during library establishment. A small sample of reverse transcribed ss- or ds-cDNA or DNA from a pre-existing library is multiplied by in vitro rolling circle ramification amplification. The resulting cDNA pseudolibrary serves as a template for numerous highly efficient PCR amplifications and permits production and analysis of target cDNAs on an automated liquid handling workstation.

CONCLUSION

The overall efficiency of the simple protocol collection approaches 100% for targets from libraries with low complexity such as Drosophila and yields >80% of amplicons up to 3 kb size in the case of human cDNA.

摘要

背景

许多结构生物学和高通量实验室都发现，从不同来源获取多个cDNA是一个相当耗时且耗费资源的过程。本文介绍的技术解决了这些问题。

结果

一种先进的靶标cDNA扩增程序，利用RNA或cDNA衍生的假文库，在文库构建过程中规避了常规的DNA转染。一小份来自预先存在文库的逆转录单链或双链cDNA或DNA，通过体外滚环分支扩增进行扩增。所得的cDNA假文库作为众多高效PCR扩增的模板，并允许在自动化液体处理工作站上生产和分析靶标cDNA。

结论

对于来自低复杂性文库（如果蝇文库）的靶标，该简单方案的总体效率接近100%；对于人类cDNA，在3 kb大小的情况下，扩增子产量超过80%。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e294/1894961/c4fbd5c407e0/1472-6750-7-31-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e294/1894961/a4478fae54cb/1472-6750-7-31-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e294/1894961/c4fbd5c407e0/1472-6750-7-31-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e294/1894961/a4478fae54cb/1472-6750-7-31-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e294/1894961/c4fbd5c407e0/1472-6750-7-31-2.jpg

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2
Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems.滤纸血斑样本DNA的全基因组扩增：对选定系统的评估
Genet Test. 2007 Spring;11(1):65-71. doi: 10.1089/gte.2006.0503.
3
Rolling circle amplification-RACE: a method for simultaneous isolation of 5' and 3' cDNA ends from amplified cDNA templates.
BMC Biotechnol. 2010 Aug 9;10:56. doi: 10.1186/1472-6750-10-56.
滚环扩增-RACE：一种从扩增的cDNA模板中同时分离5'和3' cDNA末端的方法。
Biotechniques. 2006 Jul;41(1):35-6, 38, 40 passim. doi: 10.2144/000112205.
4
Sequencing genomes from single cells by polymerase cloning.通过聚合酶克隆对单细胞基因组进行测序。
Nat Biotechnol. 2006 Jun;24(6):680-6. doi: 10.1038/nbt1214. Epub 2006 May 28.
5
Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.探索全基因组扩增作为分子遗传学研究的DNA回收工具。
J Biomol Tech. 2005 Jun;16(2):125-33.
6
Assessment of multiple displacement amplification for polymorphism discovery and haplotype determination at a highly polymorphic locus, MC1R.在高度多态性位点MC1R处，评估多重置换扩增用于多态性发现和单倍型确定的情况。
Hum Mutat. 2005 Aug;26(2):145-52. doi: 10.1002/humu.20199.
7
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Environ Microbiol. 2005 Jul;7(7):1024-8. doi: 10.1111/j.1462-2920.2005.00779.x.
8
Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification.基于phi29聚合酶的多重链置换全基因组扩增的基因组覆盖率和序列保真度。
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